Administration of plant expressed oral tolerance agents

Abstract

Protein replacement therapy for patients with hemophilia or other inherited protein deficiencies is often complicated by pathogenic antibody responses, including antibodies that neutralize the therapeutic protein or that predispose to potentially life-threatening anaphylactic reactions by formation of IgE. Using murine hemophilia B as a model, we have developed a prophylactic protocol against such responses that is non-invasive and does not include immune suppression or genetic manipulation of the patient's cells. Oral delivery of coagulation factor IX (F. IX) expressed in chloroplasts, bioencapsulated in plant cells, effectively blocked formation of inhibitory antibodies in protein replacement therapy. Inhibitor titers were mostly undetectable and up to 100-fold lower in treated mice when compared to controls. Moreover, this treatment eliminated fatal anaphylactic reactions that occurred after 4 to 6 exposures to intravenous F. IX protein. While only 20-25% of control animals survived after 6-8 F. IX doses, 90-95% of tolerized mice survived 12 injections without signs of allergy or anaphylaxis. This high-responder strain of hemophilia B mice represents the first hemophilic animal model to study anaphylactic reactions. The plant material was effective over a range of oral antigen doses (equivalent to 5-80 μg recombinant F.IX/kg), and controlled inhibitor formation and anaphylaxis long-term, up to 7 months. Oral antigen administration caused a deviant immune response that suppressed formation of IgE and inhibitory antibodies. This cost-effective and efficient approach to oral delivery of protein antigens to the gut should be applicable to several genetic diseases that are prone to pathogenic antibody responses during treatment.

What is claimed is: 
     
       1. A method for suppressing coagulation factor inhibitor formation and IgE formation in response to an intravenously administered coagulation factor in a subject, the method comprising
 expressing in a plant plastid a nucleic acid sequence encoding a coagulation factor polypeptide conjugated to cholera toxin b, and 
 orally administering to said subject a plant remnant comprising said plant plastid expressed coagulation factor conjugated to cholera toxin b, 
 wherein said oral administration can be repeated as needed to control inhibitor formation. 
 
     
     
       2. The method of  claim 1 , wherein said coagulation factor polypeptide is Factor II, Factor III, Factor IV, Factor V, Factor VI, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, or Factor XIII. 
     
     
       3. A method for suppressing coagulation factor inhibitor formation and anaphylaxis in response to intravenous administration of Factor IX to a subject receiving Factor IX treatment, the method comprising
 expressing in a plant plastid a nucleic acid sequence encoding Factor IX conjugated to cholera toxin b, and 
 orally administering to said subject a plant remnant comprising said plastid expressed Factor IX conjugated to cholera toxin b, 
 wherein a systemic antibody response against Factor IX is not induced by said oral administration, and 
 wherein said oral administration suppresses inhibitor formation and anaphylaxis in response to said intravenously administered Factor IX for up to 7 months.